Morphology of migrating telocytes and their potential role in stem cell differentiation during cartilage development in catfish (Clarias gariepinus)

Telocytes (TCs) are present in a broad range of species and regulate processes including homeostasis, tissue regeneration and immunosurveillance. This novel study describes the morphological features of migrating TCs and their role during cartilage development within the air‐breathing organ in Clarias gariepinus, the African sharptooth catfish. Light microscopy (LM), transmission electron microscopy (TEM), and immunohistochemistry (IHC) were used to examine the TCs. TCs had a cell body and telopodes which formed 3D networks in the cartilage canals and extended their telopodes to become the foremost cellular elements penetrating the cartilage matrix. The TCs were also rich in lysosomes that secreted products to the extracellular matrix (ECM). In addition, TCs formed a homocellular synaptic‐like structure that had a synaptic cleft, and the presynaptic portion consisted of a slightly expanded terminal of the telopodes which contained intermediate filaments and secretory vesicles. Gap junctions were also identified between TCs, which also connected to mesenchymal stem cells, differentiating chondrogenic cells, macrophages, apoptotic cells, and endothelial cells. In addition to describing the basic morphology of TCs, the current study also investigated migrating TCs. The TC telopodes acquired an irregular contour when migrating rather than exhibiting an extended profile. Migrating TCs additionally had ill‐defined cell bodies, condensed chromatin, thickened telopodes, and podoms which were closely attached to the cell body. The TCs also expressed markers for MMP‐9, CD117, CD34 and RhoA. In conclusion, TCs may play multiple roles during development and maturation, including promoting angiogenesis, cell migration, and regulating stem cell differentiation.

• Gap junctions form between telocytes, which also connect to mesenchymal stem cells, differentiating chondrogenic cells, macrophages, apoptotic cells, and endothelial cells.
• Migrating telocytes were discovered which had ill-defined cell bodies, condensed chromatin, thickened telopodes exhibiting irregular contours, and podoms which were closely attached to the cell body.

K E Y W O R D S
air-breathing organ, cartilage development, Clarias gariepinus, mesenchymal stem cells, migrating telocytes 1 | INTRODUCTION TCs (telocytes) are unique and multifunctional interstitial cells found in a wide range of species (Kondo & Kaestner, 2019), yet their complex and differing morphologies and functions in differing tissues means there is much to be learnt about these cells. They regulate several processes such as organogenesis, development and tissue regeneration, homeostasis (Maria-Giuliana et al., 2016;Shoshkes-Carmel et al., 2018;Zheng et al., 2013), and immunosurveillance (Jiang et al., 2018). The functions of mammalian TCs have been determined through genomic investigation (Sun et al., 2014) and proteomics analysis (Sun et al., 2014) but less is known about their morphologies and function in fish and in differing tissue types within a range of organisms. TCs have a cell body and several telopodes creating a characteristic branching profile, in turn this branching profile enables TCs to form a 3D network in the interstitium (Gherghiceanu & Popescu, 2012;Soliman & Emeish, 2017). The telopodes are distinct cellular branches that have a moniliform appearance consisting of podoms and podomeres. TCs establish long-distance cell-to-cell signaling with other cells, such as epithelial, stromal, and immune cells and are able to influence target cells through direct contact and by secretion of paracrine signaling molecules (Gherghiceanu & Popescu, 2012;Soliman & Emeish, 2017).
The current study investigated TCs during one of the vital physiological processes that occur during development, cell invasion. Cell invasion is the migration of cells that occurs during development to promote the growth of tissue. Previous studies have shown that CD117-positive mesenchymal stem cells invade the developing cartilage within embryonic and juvenile birds, mammals, and fish. These mesenchymal stem cells are driven to become chondrogenic cell lines and secrete cartilage-specific proteins (Soliman, 2014(Soliman, , 2018Soliman & Abd-Elhafeez, 2016a;. Over the last 400 million years, fish have evolved differing mechanisms to uptake atmospheric oxygen (Graham, 1997). These have included accessory respiratory organs (AROs), such as the suprabranchial chamber/organ, are anatomical structures which can assist with oxygen uptake in addition to the gills and skin. Clarias gariepinus, that are commonly called the African sharptooth catfish, developed the suprabranchial chamber/organ, a structure responsible for breathing in air, in addition to gills and skin, and is a bimodal air-breathing fish (Adamek-Urba nska et al., 2021). This fish has therefore acquired exceptional morphological-, physiological-, biochemical-and behavioral respiratory adaptations, yet relatively little is known about its AROs for an anatomical and histological overview see Mbanga et al. (2018), and the role of TCs has not been investigated. The present study examined the suprabranchial organ in catfish, as a mesenchymal-dependent mode of cartilage growth occurs within this organ, and the role of TCs during this process remains elusive.

| Ethical approval, transportation and conditions
The national ethics committee of South Valley University, Qena, Egypt (No. 88/25.10.2022) and University of Nottingham (No. 1931 170110) approved this work according to institutional, national, and international standards.
The catfish (C. gariepinus) were obtained from a private fish farm at Qena Government and transported to the aquarium laboratory at the Faculty of Veterinary Medicine, South Valley University, Qena, Egypt. The large transport water tanks were equipped with compressed oxygen, the dissolved oxygen content was 4.0-5.3 mg/L. The temperature was maintained between 26.5 and 28.9 C, with a pH of 7.2-7.9, and salinity of 12-14 ppt. All eight catfish (male and female) were healthy adults measuring 24-33 cm. Following transportation, the catfish were housed in a recirculating system in a porcelain aquarium measuring 260 Â 65 Â 70 cm. This protocol was performed according to the previously published bioassay fish protocol (Ellsaesser & Clem, 1986). The dissolved oxygen levels were maintained above 5 mg/L while water temperature was maintained at 26.5-28.9 C with a pH between 7.2 and 7.9. The fish were acclimatized to these conditions for 2 weeks and fed ad libitum on a commercial floating pellet.

| Sample collection
The present work was carried out on the air-breathing organ (suprabranchial organ) from eight catfish. All fish were anesthetized using benzocaine (4 mg/L) and decapitated. Four 1 cm 3 samples were taken for light microscopy examination and fixed in Wrobel-Mustafa fixative for 24 h (Abd-Elhafeez, Moustafa, Zayed, & Ramadan, 2017;Abd-Elhafeez, Moustafa, Zayed, & Sayed, 2017;Mnk, 2015;Wrobel & Moustafa, 2000). The remaining four samples were prepared as semithin and ultrathin samples for use in transmission electron microscopic examination and fixed with Karnovsky (1965) fixative. Table 1 shows the components of each fixative.

| Paraffin processing
Post-fixation the sample were processed as previously described (Yousef et al., 2022). The samples were washed in 70% ethanol (3 Â 24 h) to remove the fixative. They were then dehydrated in ascending grades of alcohol (80%, 90%, 100% I, 100% II), cleared in methyl benzoate, then incubated and embedded in paraffin wax for 3 h. Serial transverse and longitudinal sections 5 μm thick were prepared using a Richert Leica RM 2125 Microtome (Leica, Germany).
For general histological examination, representative sections were stained using hematoxylin and eosin (H&E) (Harris, 1900) and then set according to a previous description (Suvarna et al., 2013).

| Preparations of resin-embedded samples for semi-thin and ultrathin sections
Small specimens (0.2 mm 3 ) from the air-breathing organ were used for the semi-thin sections and were fixed in Karnovsky fixative (Karnovsky, 1965) overnight at 4 C. They were processed (Abd-Elhafeez & Soliman, 2016, 2017 as follows: Samples were washed four times for 15 min in 0.1 M sodium phosphate buffer (pH 7.2), then fixed in 1% osmic acid in 0.1 M Na-phosphate buffer for 2 h at 4 C.
Epon was a mixture of 5 mL Epon812 (Polysciences, Eppelheim, Germany) added to 5 mL araldite and 12 mL dodecenylsuccinic anhydride hardner. Samples were embedded in Epon and incubated at 60 C. The polymerization of the samples was performed by using Epon mix and accelerator (DMP30; 1.5%). The blocks were incubated for 3 days as follows: day 1 60 C, day 2 70 C, and day 3 75 C. Semithin sections (1 μm thick) were cut using an ultra-microtome Ultra cut E (Reichert-Leica, Germany) and stained with toluidine blue (Suvarna et al., 2013).

| Transmission electron microscopy (TEM)
Ultrathin sections were obtained following the semi-thin sections of air-breathing organs using a Reichert ultra-microtome. The sections (70 nm) were stained with uranyl acetate and lead citrate (Reynolds, 1963), and examined using a JEOL100CX II transmission electron microscope (Electron Microscopy Unit, Assiut University).
Digital coloration of the transmission electron microscopic images was also performed using Photoshop in order to recognize different T A B L E 1 Fixative and reagent components.

Fixative
Components Amount

| Immunohistochemistry staining (IHC)
The antibodies used had previously shown reactivity in fish and included matrix metalloproteinase-9 (MMP-9) and type II collagen and CD34, CD117 and RhoA (see Table 2) (Soliman & Abd-Elhafeez, 2016b;Soliman et al., 2019). Negative controls for all of the markers were performed using the same protocols omitting the primary antibodies.
Immunohistochemical staining was performed on paraffin sections of the air-breathing organs for matrix metalloproteinase-9 (MPP-9) and type II collagen. The paraffin-embedded 5 μm thick sections on super frost plus microscope slides were de-waxed in xylene, rehydrated in ascending grades of alcohol, and rinsed with PBS pH 7.4 (three times for 5 min).
Immunostaining was performed on semi-thin sections after removal of the resin for CD34 and CD117 (Abd-Elhafeez & Soliman, 2017; Groos et al., 2001;Lloyd, 2001) as follows: Resin sections were treated with a saturated ethanol alcoholic solution of sodium hydroxide for 5-10 min to dissolve the resin, and then the sections were rehydrated and completed according to the protocol below.
Endogenous peroxidase was suppressed using the hydrogen peroxide block at room temperature then washed with running tap water for 10 min. To enhance antigen retrieval, the slides were treated with a 10 mm sodium citrate buffer (pH 6.0) at 95-98 C in a water bath for 20 min. The sections were cooled for 20 min at room temperature and were subsequently washed in PBS (pH 7.4, three times for 5 min).
2.6 | Immunohistochemical procedures for matrix metalloproteinase-9 (MMP-9), type II collagen and RhoA Antigen localization was achieved using mouse anti-rabbit antibody against matrix MMP-9, type II collagen and RhoA (for antibody details see Table 2) using the avidin-biotin complex (ABC) technique (Hsu et al., 1981). This consisted of the following reagents: The procedure  was performed as follows: Blocking of non-specific background staining was performed using the Ultra V block for 5 min at room temperature. The primary antibodies (Table 2) were applied to different sections overnight at 4 C, then washed using PBS (at pH 7.4, three times for 5 min). The biotinylated secondary antibody was applied for 10 min at room temperature. The biotinylated secondary antibody was then applied (Table 2). Sections were washed using PBS (pH 7.4, three times for 5 min) and subsequently incubated for 10 min at room temperature in the streptavidin-peroxidase complex (Thermo Fisher Scientific, UK). Visualization of the bound antibodies was performed using one drop of DAB plus chromogen to 2 mL of DAB plus substrate.
The mixture was applied and incubated at room temperature for 5 min. The incubation processes were carried out in a humid chamber.
T A B L E 2 Antibodies used during the immunohistochemical studies. Note: All primary antibody incubations were conducted overnight, and antigen retrieval was conducted by boiling in citrate buffer (pH 6.0) for 20 min. Antibodies used showed reactivity in fish species.

| Immunohistochemical procedures for CD34 and CD117
This technique used the reagent of the DAKO En Vision TM + System, HRP peroxidase. The DAKO EnvisionTM + System, HRP is a two-step immunohistochemical staining technique (Kammerer et al., 2001;Sobhy et al., 2014).
The staining procedure (Abd-Eldayem et al., 2022) was as follows: Blocking serum (Dako) was applied for 5 min at room temperature to block non-specific background staining, followed by incubation in a primary antibody (Table 2). After incubation, the slides were washed with PBS (pH 7.4, three times Â 5 min), followed by incubation with secondary antibody (Table 2) for 30 min at room temperature. The slides were thereafter rinsed in PBS (pH 7.4, three times for 5 min) followed by incubation with 3,3 0 -diaminobenzidine (DAB) + substrate-chromogen for 5-10 min at room temperature, which resulted in a brown-colored precipitate at the antigen site.

| Immunohistochemistry counterstaining and imaging
Harris' hematoxylin was applied as counterstain for 30 s. The sections were then washed then dehydrated using 90% and 100% ethanol, then isopropanol, cleared in xylene, and covered with DPX and a cov-

| RESULTS
The air-breathing organ of the catfish was examined under light microscopy using H&E (Figure 1a-c) and toluidine blue (Figure 1d-f).
The air-breathing organ was supported by cartilage that had devel- TCs additionally formed a 3D network within the cartilage canals ( Figures 2a, 3a + b, 4a,c, and 5a). It was also noteworthy that TCs had were observed exhibiting extended telopodes which become the foremost cellular elements to penetrate the cartilage matrix (Figure 3b).
Telopodes were identified for the first time inside cartilage lacuna ( Figure 2c) and were the predominant cell population in the small cartilage canals (Figure 3b). The telopodes had aligned parallel to, and in contact with, the cartilage matrix (Figures 2c and 5a,d).
The TCs contained mitochondria (Figure 3d), endoplasmic reticulum, and were rich in lysosomes (Figure 5c-e), the content of lysosomes was secreted into the extracellular matrix (ECM) (Figures 2c-e, 3c-e, 4a-c, and 5d-f). The TCs also produced secretory vesicles which secreted products into the surrounding extracellular matrix (Figures 4b,c and 5d-f).
TCs were able to form a homocellular synaptic-like structure that This is the first study to describe the morphology of migrating TCs, and to show that their telopodes acquired an irregular contour rather than an extended profile under migratory conditions. It was also noted that migrating TCs had ill-defined cell bodies, thickened telopodes, and closely attached podoms to their cell bodies. Their cytoplasms were rich in lysosomes (Figure 5a,b,f).
The immunohistochemistry showed that TCs expressed MMP-9  (Figure 12a,b,e). Lysosomes were identified using Acridine orange where they appeared as yellow-or orange-colored vesicles located around the TCs (Figure 12c,d). The localization of TCs in the cartilage canal and relations to other cells was also illustrated ( Figure 13).

| DISCUSSION
The current study described, for the first time, the morphological features of migrating TCs. Migrating TCs in the catfish had different shapes compared with the non-migrating TCs. The overall shape, the contour of the telopodes, and the cytoplasmic organelles were the distinguishable differences between migrating and non-migrating TCs.
Non-migrating TCs had a well-defined cell body and polarized distinct telopodes, and podoms and podomeres were prominent. In contrast, migrating TCs had irregular shaped, and ill-defined, cell bodies, thickened telopodes, and podoms were closely attached to the cell body.
Acridine orange produces a metachromatic reaction that turns the lysosomes to an orange or red color (Pierzy nska-Mach et al., 2014).
The cytoplasm within migrating TCs was rich in lysosomes, as shown by AO staining, which were secreted to the ECM. We deduce that the content of lysosomes was secreted to the ECM.
The TCs expressed MMP-9 and established contact with other migratory cells such as macrophages, endothelial cells, and mesenchymal stem cells. Macrophages are wandering immune cells (Desport, 2010) while endothelial cells migrate to the developing organ to form the vascular network (Hong et al., 2010). Neural crestderived mesenchymal stem cell migration occurs in cranial and facial skeletal structures during development (Seibel et al., 2006). MMP-9 expression by the TCs may therefore facilitate the movement of macrophages and endothelial cells during development. Both MMP-2 and MMP-9 are expressed by gonad TCs in Diplectrum formosum (a fish commonly called the sand perch) and Synbranchus marmoratus (commonly termed the marbled swamp eel) (Mazzoni et al., 2019). MMP families or matrixins exert their proteolytic action on ECM components and are essential during development and tissue remodeling.
CD34 is commonly found in the hematopoietic stem cells, interstitial cells, epithelial stem cells, muscle satellite cells, corneal keratinocytes, and vascular endothelial progenitors is often expressed in TCs in a wide range of tissues from differing species including human lungs (Kondo & Kaestner, 2019;Sidney et al., 2014). In the present F I G U R E 7 Immunohistochemical staining of the air-breathing organ using CD117 on semi-thin sections. (a-c) TCs (arrowheads) as well as mesenchymal stem cells (arrow) expressed CD117. Note both the TCs and mesenchymal stem cells had cell processes. In addition, the TCs had small nuclei whilst the mesenchymal stem cells had larger nuclei.
F I G U R E 8 Immunohistochemical staining of the air-breathing organ using CD34 paraffin sections. (a, b) TCs (double arrows) expressed CD34 in the lamina propria (lp). TCs (arrows) also expressed CD34 in the perichondrium (P) in panel (b F I G U R E 1 1 Immunohistochemical staining of the air-breathing organ using RhoA on paraffin sections and negative control. (a, b) TCs (arrows) expressed RhoA in the air-breathing organ, shown alongside a negative control. study, TCs were detected expressing CD34 in the lamina propria, perichondrium, and cartilage canal. CD34, transmembrane phosphoglycoprotein, is a specific marker for TCs that has previously been identified in fish and amphibians (Mazzoni et al., 2019;Sáez et al., 2004;Zhang et al., 2016), reptiles (Gandahi et al., 2020;Yang, Ahmad, et al., 2015), birds , mammals (Zhou et al., 2015), and has now been confirmed within TCs in the catfish air-breathing organ.
The TCs in this study also expressed RhoA. RhoA is essential for cell morphology and motility regulation and is a member of the Rho family of small GTPases. Previous research has also identified RhoA as a key actin cytoskeleton regulator involved in cell shape alterations and adhesion dynamics that promote cell motility (Tkach et al., 2005).
TCs may regulate stem cell differentiation via maintenance of their microenvironment (Maria-Giuliana et al., 2016;Shoshkes-Carmel et al., 2018). TCs have also been implicated in hepatic regeneration probably through induction of hepatic stem cell proliferation after partial hepatectomy ). In the current study, CD117 positive mesenchymal stem cells were identified and additionally they had chondrogenic potential, as indicated by the expression of type II collagen. TCs were connected to both mesenchymal stem cells and differentiating chondrogenic cells, suggesting that TCs may have a role in the chondrogenic differentiation of stem cells.
The present work has shown that TCs were connected to apoptotic cells that exhibited chromatin condensation and were also rich in lysosomes. A similar finding was mentioned in the developing spinal ganglia in the quail (Coturnix coturnix japonica), indicating that TCs may regulate the apoptotic death of ganglionic cells (Soliman, 2017).
TCs have also been associated with the epithelium and stem cells in human lungs , providing an interesting comparative element between the species. Furthermore, TCs have been implicated in suppressing oxidative stress and cellular aging, and enhancing cellular renewal via inhibition of apoptosis in human lungs (Sun et al., 2014).
In conclusion, this research provides valuable insights into the structure and function of the air-breathing organ. Importantly, the present study has identified migrating and non-migrating TCs in the air-breathing organ of the catfish, and shown their differing morphological, structural and functional characteristics. These discoveries support the cross-species assertion that TCs may have multiple roles during angiogenesis promotion, apoptosis inhibition, cell migration,

CONFLICT OF INTEREST STATEMENT
The authors declare no competing interests. The authors confirm that there are no known conflicts of interest associated with this publication.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this published article and its Supplementary Information files.